Enzymatic isolation of cells and cell clusters from a biological tissue is useful for various purposes including transplantation of the cells and establishment of cell strains and in a wide variety of usages in the fields of therapy, diagnosis and examination. However, to dissociate a biological tissue and isolate cells or cell aggregates constituting the tissue, it is necessary to separate cells or cell aggregates to a desired level and isolate them from the cellular tissue. In separating cells and cell clusters from a biological tissue, the intercellular matrix is degraded by a mixture of proteases such as collagenase.
A biological tissue is constituted of cells and the intercellular matrix. The intercellular matrix, which is a substance for anchoring cells, includes structural materials and non-structural materials. The former ones include fibers such as a collagenic fiber, an elastic fiber and a reticular fiber; whereas the latter ones include so-called ground substances formed of sol- or gel-materials such as a glycoprotein and proteoglycan, for filling the space between fibers. A typical example of the intercellular matrix is a protein called collagen, which occupies about ⅓ of the weight of the total proteins in a living body. Collagen has a fiber structure, which is formally called a collagen fiber.
Tissues are roughly classified into four categories: an epithelial tissue, a supporting tissue, a muscular tissue and a nervous tissue. The epithelial tissue is a tissue covering the surface of a body, in which cells are densely arranged without the intercellular matrix interposed between them. The supporting tissue, which works for supporting organs, cells and the like, includes a connective tissue, a cartilage tissue, a bone tissue, blood and lymph. The muscular tissue is an integration of cells differentiated for purpose of contraction motion, in which the intercellular matrix occupies an extremely low ratio.
The muscular tissue is constituted of muscle cells, a connective tissue, blood vessels and nerve; however, it is primarily formed of muscle fibers. The nerve tissue is primarily constituted of the endoneurium and the perineurium, each containing a large amount of intercellular matrix (collagen). The connective tissue, which is a kind of supporting tissue, is constituted of adipose tissue and fibrous connective tissue (constituted of a collagen fiber and an elastic fiber) and roughly divided into hydrophobic connective tissue and dense connective tissue. The hydrophobic connective tissue is fibrous connective tissue having collagen fibers irregularly arranged therein and distributed in the subcutaneous tissue, mucosal tissue, nerve, outer membrane of blood vessel and interlobular tissue.
The content of collagen in the intercellular matrix varies depending upon the species, age, sex, tissue and living environment. However, it has not yet been sufficiently elucidated which type of collagen is included in which tissue and in which state of matrix in which amount. The feature of collagen resides in that amino acids constituting a peptide chain of a protein has a primary structure in which glycine repeatedly appears every third residue like “glycine-amino acid X-amino acid Y”. It has been reported that there are about beyond 30 types of collagen proteins in a human body. The collagen most abundantly present in a body is fibrous type I collagen. Non-fibrous type IV collagen is also contained abundantly and mutually connected via an intermolecular disulfide bond, contributing to formation of a reticular tissue (Non Patent Literature 1). It is reported that Type IV collagen is present between pancreatic islets and the endocrine tissue (Non Patent Literatures 2 and 3).
It may be theoretically possible to determine the presence of a predetermined type of collagen in a target matrix by immuno-staining using antibodies against individual types of collagens. However, many types of collagens are present in wide variety types of multicellular animals. Thus, it is difficult to produce antibodies against collagens. This fact serves as an obstacle and renders it difficult to realize determination of collagen by immuno-staining.
Enzymes for degrading tissue, i.e., various types of crude collagenases derived from Clostridium histolyticum contain not only two types of collagenases but also various types of proteases (having collagen degradation activity and nonspecific protein degradation activity) and non-protease components (e.g., phospholipase). By virtue of the crude collagenase, cells and cell populations are enzymatically separated from a biological tissue.
In enzymatically separating individual cells or cell populations form a biological tissue, two types of collagenases (ColG and ColH) are reported to have important roles in attaining the yield and keeping biological activity of the cells and cell populations to be separated, and thus the quantitative ratio of them has a significant effect upon the yield and activity (Non Patent Literature 4). Also, in separating pancreatic islets from the pancreatic tissue, two types of collagenases produced by Clostridium histolyticum are used (Non Patent Literature 5, Patent Literatures 1 and 2). The present inventors have so far found that pancreatic islets with high quality can be separated by optimizing the quantitative ratio of the two types of collagenases.
It has been reported that different collagenases have mutually different collagen binding domains (Non Patent Literature 6). Up to the present, various fusion proteins formed of a functional protein and a collagen binding domain have been prepared for targeting and delivery system (DDS). Examples thereof include a collagen bindable cell growth factor (Non Patent Literature 7) prepared by binding bFGF or EGF to a collagen binding domain of a collagenase derived from Clostridium histolyticum; a fusion protein formed of bovine von Willebrand factor-derived collagen binding deca-peptide and TGF-β (Non Patent Literatures 8 and 9); and a sustained-release cell growth factor supply agent (Patent Literature 3) prepared by binding a functional peptide to a collagen binding domain of fibronectin. As described above, fusion proteins with a collagen binding domain have been prepared for targeting and visualization of tissues; however, they have never been used for analysis and separation of a biological tissue.
In order to isolate a specific tissue and cells without damaging them, it is necessary to degrade the intercellular matrix present around the tissue and cells. However, it is not easy to degrade the intercellular matrix alone without degrading and damaging the surface of the desired cells. Particularly, in the case of a human organ, the proteolytic degradability varies depending upon e.g., the age, sex, habit and medical history. Thus, isolation has to be performed empirically by determining the type of enzyme and reaction time thereof.
To diabetic patients, a therapy (pancreatic islet transplantation) for transplanting pancreatic islets isolated from the pancreas is applied. For transplantation of pancreatic islets, it is essential to separate cell clumps called pancreatic islets present in the pancreatic tissue. The pancreatic tissue must be degraded without giving any damage to pancreatic islets to separate them. However, the state of the intercellular matrix significantly varies depending upon the type of animal, site of the tissue, age or sex of an individual body and growth environment. Particularly, collagen significantly changes in physical properties depending upon aging. Nevertheless, to pancreatic tissues different in state, a predetermined quantitative ratio of enzymes is applied in accordance with a protocol (except that degradation time alone is changed) and an enzymatic treatment is performed while visually checking the degree of degradation of the pancreas. For this reason, the quantity and quality of the pancreatic islets thus obtained vary depending upon the medical institution, medical workers and the state of the target pancreas.
If the type and quantity of protease to be used can be accurately and easily found from the protein composition of the extracellular matrix or organ to be degraded, target cells and the like can be isolated while maintaining high activity.